In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detection light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels back through the beam deflection device to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detection light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers, the track of the scanning light beam on or in the specimen ideally describing a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.).
German Unexamined Application DE 4330347 A1 discloses an apparatus for selection and detection of at least two spectral regions of a light beam, having a selection device and a detection device. For reliable simultaneous selection and detection of different spectral regions with high yield and with a very simple design, the apparatus is configured such that the selection device comprises means for spectral dispersion of the light beam and means on the one hand for blocking a first spectral region and on the other hand for reflecting at least a portion of the unblocked spectral region, and the detection device comprises a first detector arranged in the beam path of the blocked-out first spectral region and a second detector arranged in the beam path of the reflected spectral region. A slit diaphragm apparatus having mirror-coated diaphragm panels is preferably provided as the means for blocking a first spectral region and on the other hand for reflecting at least a portion of the unblocked spectral region. The apparatus is usable in particular as a multi-band detector in a scanning microscope.
German Unexamined Application DE 100 06 800 A1 discloses an apparatus for selection and detection of at least one spectral region of a spectrally spread-out light beam, preferably in the beam path of a confocal scanning microscope, the spread-out light beam being focusable in a focal line; and for non-overlapping detection of the spectrally spread-out light beam, the selected spectral region is characterized, in the context of an elevated number of detectors and a fault-tolerant arrangement, in that there is arranged in the spread-out light beam an optical component which reflects and/or refracts the light beam to a detector and whose optically effective region becomes smaller or larger along the surface, so that the spectral region reaching the detector is definable by means of the alignment of the component with respect to the focal line and the superposition, resulting therefrom, of the focal line and the surface.
German Unexamined Application 198 35 070 A1 discloses an arrangement for adjustable wavelength-dependent detection in a fluorescence microscope, preferably in a laser scanning microscope, comprising at least one combination, arranged in the detection beam path, of at least one short-pass filter and at least one long-pass filter to produce an adjustable bandpass, at least one filter being exchangeable with a different filter of a different wavelength characteristic and/or being adjustable in terms of its wavelength characteristic.
The arrangements disclosed in DE 4330347 A1 and DE 100 06 800 A1, which are also known by the designation “multi-band detector,” are very complex and therefore expensive. They offer the capability of multi-channel detection, which is not necessary for many applications.
The arrangement known from the aforementioned German Unexamined Application 198 35 070 A1 is not very flexible, and its handling is cumbersome. A considerable number of color filters or dichroic filters must also be kept on hand. Only discrete (but not continuous) adjustment is possible.